Printable
PDF Version
Research Article
Effect of curcumin
supplementation on blood
glucose, plasma insulin,
and glucose homeostasis
related enzyme
activities in diabetic
db/db mice
We investigated the
effect of curcumin on
insulin resistance and
glucose homeostasis in
male C57BL/
KsJ-db/db mice and their
age-matched lean
non-diabetic db/+ mice.
Both db/+ and db/db mice
were
fed with or without
curcumin (0.02%, wt/wt)
for 6 wks. Curcumin
significantly lowered
blood glucose
and HbA1c levels, and it
suppressed body weight
loss in db/db mice.
Curcumin improved
homeostasis
model assessment of
insulin resistance and
glucose tolerance, and
elevated the plasma
insulin
level in db/db mice.
Hepatic glucokinase
activity was
significantly higher in
the curcumin-supplemented
db/db group than in the
db/db group, whereas
glucose-6-phosphatase
and phosphoenolpyruvate
carboxykinase activities
were significantly
lower. In db/db mice,
curcumin significantly
lowered
the hepatic activities
of fatty acid synthase,
b-oxidation,
3-hydroxy-3-methylglutaryl
coenzyme
reductase, and acyl-CoA:
cholesterol
acyltransferase.
Curcumin significantly
lowered plasma free
fatty acid, cholesterol,
and triglyceride
concentrations and
increased the hepatic
glycogen and skeletal
muscle lipoprotein
lipase in db/db mice.
Curcumin normalized
erythrocyte and hepatic
antioxidant
enzyme activities
(superoxide dismutase,
catalase, gluthathione
peroxidase) in db/db
mice that
resulted in a
significant reduction in
lipid peroxidation.
However, curcumin showed
no effect on the
blood glucose, plasma
insulin, and glucose
regulating enzyme
activities in db/+ mice.
These results
suggest that curcumin
seemed to be a potential
glucose-lowering agent
and antioxidant in type
2 diabetic
db/db mice, but had no
affect in non-diabetic
db/+ mice.
Keywords: Antioxidant /
Curcumin / Glucose
homeostasis / Insulin
resistance / Type 2
diabetes /
1 Introduction
Curcumin
[1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadien-
3,5-dione] is the active
component in Turmeric
Rhizomes
(Curcuma Long Linn),
which are the major
component
of spices turmeric and
curry. These spices have
been
widely used in
traditional medicine in
Southeast Asia, and
their numerous
biological effects have
been associated with
curcumin [1].
Practitioners of
traditional Indian
medicine
believe that curcumin
powder prevents many
diseases
including biliary
disorders, anorexia,
cough, diabetes, hepatic
disorders, rheumatism,
sinusitis, cancer, and
Alzheimer's
[2]. Several studies
have indicated that
curcumin
plays a beneficial role
in terms of being an
antioxidant,
anti-tumurogenic, and
anti-inflammatory agent
[3].
A recent study showed
that curcumin-treated
diabetic
rats had lower blood
glucose and glycated
hemoglobin
levels, in association
with lower oxidative
stress [4].
Furthermore,
treatment with curcumin
has been shown to
reduce reactive oxygen
species (ROS) levels in
cells that
are isolated from
diabetic patients [5].
Experimental studies
with diabetic animals
demonstrated that
curcumin supplementation
can suppress cataract
development [6] and the
cross-linking of
collagen [7], promotes
wound healing [8],
and lower blood lipid
and glucose levels in a
streptozotocintreated
diabetic animal model
[9].
Glucose homeostasis is
regulated primarily by
the liver
and skeletal muscle.
Following a meal, most
glucose disposal
occurs in skeletal
muscle, whereas fasting
plasma glucose
levels are determined
primarily by a glucose
output
from the liver [10]. In
particular, hepatic
insulin resistance
is associated with
increased free fatty
acid (FFA) that lowers
the ability of insulin
to suppress hepatic
glucose production
by activating
gluconeogenesis yet
inhibiting glycolysis
[11]. Therefore, this
study was to examine the
effect of curcumin
on insulin resistance,
glucose homeostasis, and
oxidative
damage in
C57BL/KsJ-db/db mice, a
good model for
type 2 diabetes that
displays many of the
characteristics of
human disease including
hyperphagia,
hyperglycemia,
insulin resistance, and
progressive obesity
[12].
2 Materials and methods
2.1 Animals and diets
Sixteen male C57BL/KsJ
db/db mice and sixteen
lean heterozygote
non-diabetic db/+ were
purchased from Jackson
Laboratory (Bar Harbor,
ME). The animals were
individually
housed in stainless
steel cages in a room at
22 l 28C on
a 12 h light-dark cycle.
The five week old db/db
mice and
db/+ mice were fed a
pelletized commercial
chow diet for
2 wks after arrival,
then the db/+ and db/db
mice were randomly
divided into two groups
(n=8), respectively.
Thereafter,
both db/+ mice and db/db
mice were fed a standard
semisynthetic diet
(AIN-76) [13, 14] with
curcumin (0.2 g/
kg diet, Sigma) or
without for 6 wks. The
mice had access
to food and water ad
libitum. At the end of
the experimental
period, the mice were
anesthetized with
Ketamine after
withholding food for 12
h, and blood samples
were taken
from the inferior vena
cava to determine the
plasma biomarkers.
Also, the liver was
removed after collecting
the
blood, and rinsed with a
physiological saline
solution. All
mice were treated in
accordance with Sunchon
National
University Guidelines
for the Care and Use of
Laboratory
Animals.
2.2 Blood glucose and
glycosylated hemoglobin
(HbA1c) concentrations
The blood glucose
concentration was
monitored in venous
blood drawn from the
tail vein using a
glucometer (Allmeicus,
Korea) every week after
a 6 h fast. The blood
HbA1c
concentration was
measured after hemolysis
of the anticoagulated
whole blood specimen.
HbA1c was determined
immuno-turbidinetrically.
2.3 Intraperitoneal
glucose tolerance test
(IPGTT)
An IPGTTwas performed at
the fifth week.
Following a 6 h
fast, the mice were
injected
intraperitoneally with
glucose
at 1 g/kg body weight,
and the blood glucose
levels were
determined in tail blood
samples taken 0 (prior
to glucose
administration), 30, 60,
and 120 min after the
glucose
administration.
2.4 Homeostatic index of
insulin resistance
(HOMA-IR)
HOMA-IR was calculated
according to the
homeostasis of
the assessment as
follows (Eq. 1) [15]:
HOMA-IR = [fasting
glucose (mmol/L)
6fasting insulin (lL
U/mL)]/22.51
2.5 Plasma biomarkers
Plasma insulin
(Diagnostic System
Laboratories, USA) and
leptin (Linco, USA)
levels were determined
using radioimmunoassay
kits. The plasma total
cholesterol and
triglyceride
concentrations were
determined using an
enzymatic
method (Sigma), while
the plasma FFA
concentration was
determined using an
enzymatic colorimetric
method (Wako
Chemicals, Richmond,
VA).
2.6 Sample preparation
Blood samples were
collected from the
inferior vena cava
using heparin-coated
tubes. After
centrifugation at
10006g for 15 mim at
48C, the plasma and
buffy coat were
carefully removed. The
separated cells were
then washed
three times by
resuspension in a 0.9%
NaCl solution and
the centrifugation was
repeated. The washed
cells were
lyzed in an equal volume
of water and mixed
thoroughly.
The hemoglobin
concentration was
estimated in an aliquot
of the hemolysate, using
a commercial assay kit
(No. 525-
A, Sigma). An
appropriate dilution of
the hemolysate was
then prepared from the
erythrocytes suspension
by the addition
of distilled water to
estimate the catalase
(CAT) and
glutathione peroxidase
(GSH-Px) activities.
In addition, to remove
the hemoglobin by
precipitation
with chloroform:ethanol
[16], 0.2 mL of an
ethanol:choloroform
(3:5, v/v) mixture was
added to an aliquiot
(0.5 mL) of the
hemolysate cooled in
ice. This mixture was
stirred constantly for
15 min and then diluted
with 0.1 mL
of water. After
centrifugation for 10
min at 16006g, the
pale yellow supernatant
was separated from the
protein precipitate
and used to assay
superoxide dismutase
(SOD).
The enzyme source
fraction in the liver
was prepared
according to the method
developed by Hulcher and
Oleson [17] with slight
modifications. A 20% w/v
homogenate was
prepared in a buffer
containing 0.1 mol/L
triethanolamine,
0.02 mol/L EDTA and 2
mmol/L DTT (pH 7.0),
then centrifuged
at 6006g for 10 min to
discard any cell debris,
and
the supernatant
centrifuged at 100006g
followed by
120006g for 20 min at
48C to remove
mitochondrial pellets.
Thereafter, the
supernatant was
ultracentrifuged twice
at 1000006g for 60 min
at 48C to obtain the
cytosolic
supernatant. The
mitochondrial and
microsomal pellets
were then redissolved in
800 lL of a
homogenization buffer
and the protein content
was determined by method
of Bradford
[18] using BSA as the
standard.
2.7 Hepatic glucose
regulating enzyme
activities
and glycogen content
Glucokinase (GK)
activity was determined
using a
spectrophotometric
continuous assay as
described by Davidson
and Arion [19] and
Newgard et al. [20] with
slight modifications,
where the formation of
glucose-6-phosphate was
coupled to its oxidation
by glucose-6-phosphate
dehydrogenase
and NAD+ at 378C.
Glucose-6-phosphatase
(G6Pase) activity was
determined using the
method of Alegre
et al. [21] with slight
modifications; the
reaction mixture
contained 40 mmol/L of
sodium HEPES (pH 6.5),
14 mmol/L of
glucose-6-phosphate, 18
mmol/L of EDTA,
both previously adjusted
to pH 6.5, 2 mmol/L of
NADP+,
0.6 IU/mL of mutarotase
and 0.6 IU/mL of glucose
dehydrogenase.
Phosphoenolpyruvate
carboxykinase (PEPCK)
activity was monitored
in the direction of
oxaloacetate synthesis
using the
spectrophotometric assay
developed by
Bentle and Lardy [22]
with slight
modifications. The final
volume of the purified
enzyme (1 mL) was
pipetted with a
reaction mixture (pH
7.0) containing; 77
mmol/L sodium
HEPES, 1 mmol/L inosine
59-diphosphate, 1 mmol/L
MnCl2, 1 mmol/L DTT,
0.25 mmol/L NADH, 2
mmol/L
phosphoenolpyruvate, 50
mmol/L of NaHCO3 and 7.2
U
malic dehydrogenase. The
enzyme activity was then
measured
for 2 min at 258C based
on a decrease in the
absorbance
at 340 nm.
Hepatic glycogen
concentration was
determined as
described previously by
Seifter et al. [23] with
modifications.
The liver tissue was
homogenized in five
volumes of
a 30% w/v KOH solution
and dissolved at 1008C
for
30 min. The glycogen was
determined by treatment
with an
anthrone reagent and
measuring the absorbance
at 620 nm.
2.8 Hepatic lipid
regulating enzyme
activities
Fatty acid synthase
(FAS) activity was
determined as
described by Nepokroeff
et al. [24] with slight
modifications.
The cytosolic enzyme
(100 lL) was mixed with
125 mmol/L of potassium
phosphate buffer (pH
7.0),
165 lmol/L of
acetyl-CoA, 50 lmol/L of
malonyl-CoA,
50 lmol/L of NADPH, 1
mmol/L of
b-mercaptoethanol
and 1 mmol/L EDTA.
Absorbance was then
measured for
2 min at 340 nm (308C)
on a spectrophotometer.
b-Oxidation
activity was determined
as described by Lazarow
[25]
with slight
modifications, where the
reaction was initiated
by adding 47 mmol/L of
Tris-HCl (pH 8.0), 0.2
mmol/L of
NAD, 990 lmol/L DTT, 5
lL albumin (1.5%), 5 lL
Triton
X-100 (2%), 0.1 mmol/L
CoA, 0.01 mmol/L FAD, 1
mmol/
L KCN and 5 lL of the
mitochondrial fraction,
then
10 lmol/L palmitoyl-CoA
was added. The formation
of
NADH was measured for 5
min at 340 nm (378C) on
a spectrophotometer.
The carnitine
palmitoyltransferase
(CPT)
was assayed
spectrophotometrically
by following the
release of CoA-SH from
palmitoyl-CoA using the
general
thiol reagent
5,59-dithiobis
(2-nitrobenzoate), DTNB,
as
described by Bieber et
al. [26] with slight
modifications.
The reaction mixture
contained 0.1 mL aliquot
of a premix
containing 232 mmol/L
Tris-HCl (pH 8.0), 1.1
mmol/L
EDTA, 220 lmol/L
L-carnitine, 24 lmol/L
of DTNB,
7 lmol/L palmitoyl-CoA
and 0.09% Triton X-100.
The
reaction was initiated
by the addition of
enzyme at 258C.
Absorbance was measured
for 2 min at 412 nm on a
spectrophotometer.
The HMG-CoA reductase
activities were
determined in the
microsomal fraction with
[14C]-HMGCoA
as the substrate based
on a modification of the
method
of Shapiro et al. [27].
The activity was
expressed as the
synthesized
mevalonate pmol/min/mg
protein. The acyl-
CoA:cholesterol
acyltransferase (ACAT)
activities were
determined by the rate
of the incorporation of
[14C]-Oleoyl
CoA into cholesterol
ester fractions, as
described by Erickson
et al. [28] and modified
by Gillies et al. [29].
The activity
was expressed as
synthesized
cholesteryloleate pmol/
min/mg protein.
2.9 Lipoprotein lipase
(LPL) activity in
adipose
tissue and skeletal
muscle
A 10% w/v homogenate was
prepared in a
detergent-containing
buffer (25 mmol/L
ammonium chloride, 5
mmol/L
EDTA, 10 mg/mL Triton
X-100, 1 mg/mL SDS, 5
IU/mL
heparin, 10 lg/mL
leupeptin, 1 lg/mL
pepstatin A, 3.5 lg/
mL aprotinin, pH 8.5),
then centrifuged at
200006g (48C)
for 20 min to obtain the
supernatant. Thereafter,
LPL activity
was determined according
to the method developed
by
Nilsson-Ehle and Schotz
[30] with slight
modifications. To
prepare the substrate,
600 mg of triolein, 36
mg of phosphatidyl
cholin (egg yolk, Sigma)
and 3H-triolein (2.56109
dpm, specific activity;
22 Ci/mmol) were added
and dried
in N2 gas. The dried
substrate was then added
to 10 mL of
glycerol and sonicated.
The prepared substrate,
heat-inactivated
serum (at 568C for 30
min), and a 0.3 mol/L of
Tris-
HCl (pH 8.5) buffer
containing 0.2 mol/L
NaCl, 0.02% w/v
heparin and 12% w/v
BSAwere then used for
the assay mixture
and 120 lL preincubated
at 378C for 5 min. Next,
the
reaction was initiated
by adding 5 lL of the
tissue preparations
and 75 lL of distilled
water to make a final
volume of 0.2 mL, which
was then heated at 378C
for 1 h. The FFA
that formed was isolated
using a liquid-liquid
partition system
[31]. Thereafter, the
reaction was stopped by
the addition
of 3.25 mL of
methanol:chloroform:heptane
(1.41:1.25:1, v:v:v) and
1.05 mL of a 0.1 mol/L
sodium carbonate
buffer (pH 10), then the
reaction mixture was
centrifuged
for 15 min at 15006g.
Finally, 0.4 mL of the
supernatant
was subjected to
scintillation counting
(Packard Tricarb
1600TR, Packard,
Australia).
2.10 Antioxidant enzyme
activities
SOD activity was
spectrophotometrically
measured using a
modified version of the
method developed by
Marklund
and Marklund [32].
Briefly, SOD was
detected on the basis
of its ability to
inhibit
superoxide-mediated
reduction. One
unit was determined as
the amount of enzyme
that inhibited
the oxidation of
pyrogallol by 50%.
CATactivity was measured
using Aebi's [33] method
with slight
modifications, in
which the disappearance
of hydrogen peroxide was
monitored
spectrophotometrically
at 240 nm for 5 min. A
molar
extinction coefficient
of 0.041 mM– 1cm– 1 was
used to
determine CAT activity.
The GSH-Px activity was
measured
using Paglia and
Valentine's [34] method
with slight
modifications. The
reaction mixture
contained 1 mmol/L
glutathione, 0.2 mmol/L
NADPH, and 0.24 units of
glutathione
reductase in a 0.1 mol/L
Tris-HCl (pH 7.2)
buffer.
The reaction was
initiated by adding 0.25
mmol/L H2O2
and the absorbance was
measured at 340 nm for 5
min. A
molar extinction
coefficient of 6.22 mM–
1cm– 1was used to
determine the activity.
2.11 Lipid peroxidation
As a marker of lipid
peroxidation production,
the erythrocyte
or hepatic
malondialdehyde (MDA)
concentrations
were measured using the
method of Ohkawa et al.
[35]. Two
hundred microliters of
the erythrocyte and
hepatic homogenate
(20%, w/v) were mixed
with 200 lL of 8.1% w/v
SDS,
1.5 mL of 20% w/v acetic
acid (pH 3.5), and 1.5
mL of
0.8% w/v thiobarbituric
acid. The reaction
mixture was
then heated at 958C for
60 min. After cooling,
the hepatic
mixture was added to 1.0
mL of distilled H2O and
5.0 mL
of a butanol:pyridine
(15:1) solution. The
reaction mixture
was then centrifuged at
8006g for 15 min and the
resulting
colored layer was
measured at 532 nm using
1,1,3,3-tetraethoxypropane
(Sigma) as the standard.
2.12 Statistical
analysis
All data are presented
as the mean l SE.
Statistical analyses
were performed using the
SPSS program (SPSS,
Chicago,
IL). Student's t-test
was used to assess the
differences
between the groups. The
db/+ group was compared
with the
db/+ curcumin, db/db,
and db/db curcmin
groups. The
effect of the curcumin
supplement was also
compared
within the type 2
diabetic mice groups.
Values of p a 0.05
were considered to be
statistically
significant.
3 Results
3.1 Body weight and food
intake
As shown in Table 1,
body weights in the
db/db groups were
significantly higher
than in the non-diabetic
db/+ groups.
We observed that the
body weight reduction of
db/db mice
was significant at 6 wks
(30.80 l 1.24 g vs.
33.03 l 1.08 g,
p a 0.05). Curcumin
supplement did not
affect body weight
of db/+ mice, however it
suppressed a reduction
in body
weight within the db/db
groups. The final body
weight of
the
curcumin-supplemented
mice was significantly
higher
compared to the db/db
group by 1.5-fold (Table
1). During
the experimental period,
the food intake in the
db/db groups
were also higher than in
the db/+ groups, however
curcumin
did not affect the food
intake in both db/+ and
db/db groups
(data not shown).
3.2 Fasting blood
glucose level and IPGTT
Baseline (0 wk) fasting
glucose levels did not
differ
between the groups,
however the blood
glucose values of
the db/db groups were
significantly higher
than those of db/
+ groups during the six
week testing period
(Fig. 1). Curcumin
supplement did not
change blood glucose
level in nondiabetic
group, however it
significantly lowered
blood glucose
level in diabetic db/db
group starting from the
first
week. This resulted in
lowering the glucose
level by 22% in
the db/db mice at the
sixth week (Fig. 1).
Although we also
did not observe change
of IPGTT in db/+ mice,
curcumin
significantly improved
the glucose tolerance in
db/db
groups over the entire
IPGTT (Fig. 2). As such,
the curcu-
4i 2008 WILEY-VCH Verlag
GmbH & Co. KGaA,
Weinheim
www.mnf-journal.com
Figure 1. Blood glucose
level in db/db mice and
db/+ mice
fed diet supplemented
with curcumin. Values
are expressed
as mean l SE, a p a 0.05
vs. to db/+ group based
on Student's
t-test, # p a 0.05 vs.
to db/db group based on
Student's t-test. min
improved fasting blood
glucose and postprandial
glucose
level in only type 2
diabetic mice.
3.3 Blood HbA1c
concentration, plasma
biomarkers, and HOMA-IR
Blood HbA1c
concentrations in the
db/db groups were
approximately 3-fold
higher than in the
non-diabetic db/+
groups, however the
curcumin supplement
significantly
lowered the blood HbA1c
concentration within the
db/db
mice groups by 7.8%
(Table 2). The plasma
leptin concentrations
were significantly
higher in the db/db
groups than
in the non-diabetic db/+
groups. The curcumin
supplementation
within the db/db groups
elevated the leptin
concentration
compared to the diabetic
control db/db group by
1.6-
fold (Table 2). The
plasma insulin level of
the curcuminsupplemented
db/db mice was
significantly higher
than in
the control db/db mice
by 17% (Table 2).
HOMA-IR in the
db/db groups was
significantly higher
than in the db/+
groups by 7-fold,
however curcumin
significantly improved
HOMA-IR in db/db mice
(Table 2). No
differences were
observed within the
non-diabetic db/+
groups.
3.4 Plasma lipid levels
Plasma fatty acid, total
cholesterol, and
triglyceride
concentrations
were significantly
higher in the db/db
group than in
the db/+ group, however
the curcumin supplement
significantly
lowered these lipid
profiles by 17, 19, and
10%,
respectively, within the
type 2 diabetic mice
groups (Table
2). No differences were
observed within the
non-diabetic
db/+ groups.
3.5 Hepatic glucose
regulating enzyme
activities
and glycogen level
Hepatic GK activity was
significantly lower in
the db/db
group than in the db/+
group, yet the G6Pase
and PEPCK
activities were
significantly higher in
the db/db groups
compared to the db/+
groups (Table 3).
Curcumin supplement
significantly elevated
hepatic GK activity
within the
db/db groups by 42%,
while it significantly
suppressed the
elevation of hepatic
gluconeogenic enzyme
activities,
G6Pase and PEPCK, in
db/db mice (Table 3).
The hepatic
glycogen concentration
in the db/db group was
about 2.5
times higher than in the
db/+ group. The curcumin
supplement
significantly elevated
glycogen storage in the
liver
within the db/db groups
(Table 3). In
non-diabetic db/+
mice, curcumin did not
alter hepatic GK,
P6Pase, and
PEPCK activities and
glycogen content.
3.6 Hepatic lipid
regulating enzyme
activities
The activities of
hepatic FAS,
b-oxidation, CPT, HMGCoA
reductase, and ACATwere
significantly higher in
the
db/db group than in the
non-diabetic db/+ group
(Table 4).
The elevated hepatic
lipid regulating enzymes
activities
were significantly lower
in the
curcumin-supplemented
group compared to the
db/db group (Table 4).
No differences
were observed within the
non-diabetic db/+
groups. 3.7 LPL
activities in adipose
tissue and skeletal
muscle
The LPL activities of
adipose tissue were
significantly
lower in the db/db
groups than in the
non-diabetic db/+
groups, whereas, those
of the skeletal muscle
were significantly
higher in the db/db
groups (Fig. 3).
Although curcumin
did not affect adipose
tissue LPL activity,
skeletal
muscle LPL activity was
significantly higher in
the curcumin-
supplemented db/db mice
than in the db/db groups
(Fig. 3). No differences
were observed within the
non-diabetic
db/+ groups.
3.8 Antioxidant defense
enzyme activities and
lipid peroxide in the
erythrocytes and liver
Erythrocyte SOD and CAT
activities were
significantly
higher in the db/db
group than in the db/+
group, but the
GSH-Px activity was
lower (Table 5).
Supplementation
with curcumin lowered
the erythrocyte SOD and
CATactivities
and elevated GSH-Px
activity in db/db
groups. Hepatic
SOD activity did not
differ between the
groups, however
CAT and GSH-Px
activities were higher
in the db/db group
than in the db/+ group.
These increased CAT and
GSH-Px
activities were
significantly lowered by
the curcumin supplement
when compared within the
db/db groups (Table 5).
As such curcumin seemed
to normalize altered
erythrocyte
and hepatic antioxidant
enzyme activities within
the db/db
mice. Erythrocyte and
hepatic MAD levels,
index of lipid
peroxide, were
significantly higher in
db/db group than in
the db/+ group by
1.5-fold and 1.8-fold,
respectively (Table
5). However, curcumin
effectively lowered MDA
levels in 4 Discussion
This study demonstrates
that the dietary
curcumin supplement
improved insulin
resistance and
hyperglycemia in db/
db mice, obese-diabetic
animals with insulin
resistance, but
had no effects on
non-diabetic db/+ mice.
In the present
study, curcumin
significantly lowered
blood glucose levels
and HOMA-IR when
compared to those in the
diabetic control
db/db mice by 22 and
10%, respectively.
HOMA-IR has
been suggested as a
biomarker to assess
insulin resistance
and secretion and is a
useful clinical index
for insulin sensitivity
and pancreatic b-cell
functions in
epidemiological
studies [15]. Although
HOMA-IR has several
limitations in
terms of accuracy and
reliability [36], it
expresses essentially
hepatic insulin
resistance [37]. We also
observed curcumin
significantly improved
the glucose tolerance
within
db/db groups without
changing of IPGTT in
db/+ mice.
The db/db mouse, which
has a mutation in the
leptin
receptor gene that
caused abnormal
splicing, acquires
obesity
and develops type 2
diabetes as a
consequence of loss
of leptin function [38].
This type of genetic
control over the
pathogenetic mechanisms
can lead to insufficient
insulin
secretion [39]. These
results show that
curcumin is able to
improve insulin
resistance with a
simultaneous increase in
plasma leptin and
insulin levels. The
plasma insulin levels
of db/db mice increase
rapidly during the first
weeks of life,
but they dramatically
decrease at an age 10–12
wks to normal
or less than normal
levels [40], resulting
in drastic body
weight loss at the time
of death [12]. However,
the curcumin
supplement suppressed
body weight loss in
db/db mice by
sustaining plasma leptin
and insulin levels.
Curcumin
increased plasma leptin
and insulin levels by 60
and 17% in
db/db groups,
respectively, however it
did not affect those
of db/+ mice. Although,
we can not provide
direct evidence
for the effect of
curcumin on insulin
release in db/db mice,
recently curcumin was
reported to enhance
insulin release
by induction of b-cell
electrical activity
[41]. In our previous
studies, plasma insulin
levels showed a positive
correlation
with leptin levels in
db/db mice [42, 43].
Insulin induces
leptin synthesis and
secretion [44] through
the regulation
of glucose metabolism in
adipocytes [45]. The
exact
relationship between
increased leptin levels
and decreased
blood glucose level
could not be clarified,
however we observed a
negative correlation
between plasma leptin
and
blood glucose
concentration (r =
–0.622, p a 0.01) within
db/db mice. Harris et
al. [46] reported that
chronic intraperitonal
leptin infusion (7 days)
significantly reduced
fasting
glucose in male
C57BL/Ks-db/db mice, by
suggesting that
it is mediated by leptin
receptors other than the
hypothalamic
long-form receptors.
However, no clear
explanation is
available at this
moment.
Insulin resistance
profoundly contributes
to the pathophysiology
of type 2 diabetes and
it reduces glucose
utilization
and increases glucose
production from the
liver, thus
leading to hyperglycemia
[47]. The liver plays a
unique role
in controlling
carbohydrate metabolism
by maintaining a
normal range of glucose
concentration. Knowledge
of the
processes involved in
maintaining glucose
homeostasis as
well as insulin
resistance is a
prerequisite for
developing
new therapeutic approach
in diabetes as well as
in liver disease
[48]. Our results show
that curcumin influences
the
glucose and lipid
metabolism in the liver
as well as in
muscle. We observed
hepatic GK activity was
significantly
lower in the db/db mice
than in the db/+ mice,
whereas glycogen
content of db/db mice
was higher compared to
the
db/+ mice. These
findings contrast with
those of previous
reports by Yen and Stamm
[49] who showed that GK
activity
was higher in the db/db
mice than in the control
mice,
and by Roesler and
Khandelwal [50] who
reported little
difference
in hepatic glycogen
levels between db/db and
db/+
mice. However, Coleman
and Hummel [51] reported
glycogen
level was 2–3-fold
higher in diabetic mice
relative to
control. In db/db mice,
curcumin increased
hepatic GK
activity and glycogen
content, while it
inhibited G6Pase
and PEPCK activities. In
our previous studies, we
also
found that similar
amounts of various
flavonoid compounds
increased GK activity
and its mRNA expression
and lowered
G6Pase and PEPCK
activities and their
mRNA expression
in the liver of db/db
mice [42, 52]. These
results suggest
that down-regulation of
gluconeogenic enzymes
and
the up-regulation of
glycolytic enzymes by
curcumin have
contributed towards
reduced blood glucose
concentration in
curcumin-supplemented
db/db mice.
Prolonged exposure to
high fatty acid
concentrations
(lipotoxicity)
influences both insulin
action and insulin
secretion, and it can
contribute directly to
the deterioration
of pancreatic b-cell
functions that
accompanies the
development
of diabetes [53]. In our
study, the plasma FFA
and
triglyceride
concentrations were
significantly higher in
the
db/db group than in the
db/+ group by 2.2-fold
and 1.5-
fold, however the
curcumin supplement
lowered those of
db/db mice. LPL is a
rate-limiting enzyme
that hydrolyzes
circulating
triglyceride, leading to
the generation of FFA,
which store triglyceride
in adipose tissue and
serve energy
source in skeletal
muscle and heart [54].
Impaired insulin
action not only
stimulates lipolysis,
increasing delivery of
FFA to the liver and
consequently increasing
production of
hepatic triglyceride but
also reduces LPL
activity [55]. We
also observed similar
results that the LPL
activity in adipose
tissue was significantly
lower in the diabetic
db/db
group than in the
non-diabetic db/+ group,
but hepatic triglyceride
concentration was
significantly higher in
the diabetic
db/db group (13.69 l
0.40 mg/g vs. 10.86 l
0.12 mg/
g, p a 0.05).
Interestingly, curcumin
did not affect the LPL
activity in adipose
tissue and hepatic
triglyceride
concentration
in db/db mice (data not
shown). The LPL activity
in
skeletal muscle was
significantly higher in
the diabetic db/
db group than in the
non-diabetic db/+ group.
This muscle
LPL activity was even
further elevated by
curcumin supplement
in db/db group. Our
results are consistent
with other's
report that the
activation of LPL in
adipose tissue is
delayed,
whereas LPL activity in
skeletal muscle is
increased by
hyperinsulinemia in
obesity and type 2
diabetes [56]. Somehow,
curcumin supplement only
elevated LPL activity in
skeletal muscle of db/db
mice. This appeared to
contribute
to lower plasma
triglyceride
concentration in
curcuminsupplemented
db/db mice, although
further study is
required to support the
action of curcumin on
altering the
LPL activity in skeletal
muscle. Furthermore,
insulin resistance
is commonly associated
with several
abnormalities in
lipid metabolism,
including increased
plasma fatty acid
levels,
hypertriglycemia,
hypercholesterolemia and
enhanced
hepatic lipogenesis. In
the current study, we
observed that
the hepatic FAS,
b-oxidation, CPT,
HMG-CoA reductase
and ACAT activities were
significantly higher in
the db/db
mice than in the db/+
mice, however the
supplementation
of curcumin significant
lowered these lipid
regulating
enzyme activities in
db/db mice. Curcumin
significantly
lowered plasma FFA and
cholesterol
concentrations by
altering their metabolic
enzyme activities.
Hyperglycemia increases
oxidative stress through
the
overproduction of ROS
[57]. These ROS
contribute to
organ injury in systems
such as the heart and
liver [58], and
oxidative damage is
generally increased in
diabetes [59]. In
particular, erythrocytes
are susceptible to
oxidative damage
resulting from a high
concentration of oxygen
and hemoglobin
[60]. In the present
study, erythrocyte SOD
and CAT
activities were
significantly higher in
the db/db control
group than in the db/+
control group, whereas
GSH-Px
activity was lower in
the db/db group. Ahmed
et al. [61]
also reported that serum
SOD and CAT activities
were significantly
higher in type 2
diabetic patients as
compared to
the control. These
antioxidant enzymes were
also suggested
as being markers for
vascular injury in type
2 diabetes [61].
The hepatic SOD activity
did not differ between
db/db
group and db/+ group,
whereas CAT and GSH-Px
activities
were significantly
increased in db/db mice.
In the current
study, curcumin
normalized the
antioxidant enzymes
activities
of erythrocyte and liver
in db/db mice. It is
plausible
that curcumin
supplementation seemed
to alter these
enzymes activities in
erythrocyte and liver
toward maintaining
antioxidant homeostasis
in db/db mice. Elevated
glucose levels induce
oxidative stress that is
ultimately reflected by
the increased MDA levels
in the erythrocytes
and liver of db/db mice.
Lipid peroxidation is
also considered
responsible for the
impairment of
endothelial cells,
capillary permeability,
and fibroblast and
collagen metabolism
[62]. In this study,
curcumin significantly
lowered
MDA levels in the
erythrocytes and liver
of db/db mice,
thus indicating a
decreased rate of lipid
peroxidation. As
such curcumin may
decrease the production
of free radicals
which could lead to
normalizing antioxidant
activity in the
erythrocytes and liver.
In conclusion, these
results suggest that
curcumin was
beneficial in improving
insulin resistance and
glucose
homeostasis in db/db
mice, which seems to be
medicated
through elevation of
plasma insulin level
that caused activation
of glycolysis and
inhibition of
gluconeogenic and lipid
metabolic enzymes in
liver, and increasing
LPL activity in
skeletal muscle,
although further
detailed mechanism needs
to be elucidated. In
addition, curcumin
seemingly contributed
to a reduction in
oxidative stress that
was induced by
hyperglycemia in the
erythrocytes and liver,
thereby being
beneficial in preventing
diabetic complication.
This work was supported
by the NURI project of
the Ministry
of Education, Korea in
2006.
Printable
PDF Version
